The effects of nicotine on the time-course of the secretion of [Met5]-enekephalin (ME) in addition to proenekephlin A (proENK) mRNA levels, and on the transcriptional activity of the proENK gene were studied in bovine adrenal medullary chromaffin (BAMC) cells. Nicotine at a concentration of 10 ~M caused a rapid secretion (within one hour) of ME followed by a long-term secretion (12-24 hours after treatment) into the medium. Post-treatment with the cholinergic antagonists, hexamethonium (1 mM) and atropone (1~M), up to six hours after the nicotine treatment significantly inhibited the long-term secretion of ME induced by nicotine without affecting the short-term secretion. However, nicotine-induced long-term secretion of ME was not affected when cholinergic antagonists were added 9 or 12 hours after the nicotine treatment. Long -term (24 hour) stimulation of BAMC cells with nicotine also increased proENK mRNA levels by about 2-fold. This nicotine-induced response was inhibited by post-treatment with cholinergic antagonist 0.5,1,3,and 6, hours after the nicotine treatment. Like the secretion experiments, these cholinergic antagonists did not affect the nicotine-induced response when they were given after 9 and 12 hours. Similar to cholinergic antagonists studies, post-treatment with nimodipine (1 ~M) or calmidazolium (1 ~M) up to six hours after the nicotine treatment significantly inhibited the increase of the long-term secretion of ME and proENK mRNA levels induced by nicotine. However, they were ineffective in blocking the long-term secretion of ME and proENK mRNA levels induced by nicotine when BAMC cells were post-treated after 9 and 12 hours. The results of the nuclear run-on assay showed that nicotine increases the transcriptional rate for the proENK gene after 30 min and the response continues for up to nine hours with a maximal increase of 2-to 2.5-fold. Our results suggest that the long-term stimulation (for at least six hours) of nicotinic receptors is required for the increases in proENK mRNA levels and the long-term secretion of ME in BAMC cells. The nicotine-induced long-term secretion of ME followed an increased biosynthesis of ME during the first six hours which appeared to result from increased transcription of the proENK gene followed by increased synthesis of proENK mRNA.